Download Macular pigments: their characteristics and putative role

ARTICLE IN PRESS
Progress in Retinal and Eye Research 23 (2004) 533–559
Macular pigments: their characteristics and putative role
Nigel P. Daviesa, Antony B. Morlandb,*
a
Department of Ophthalmology, Chelsea and Westminster Hospital, Fulham Road, London SW10 9NH, UK
b
Psychology Department, Royal Holloway University of London, Egham, Surrey TW20 0EX, UK
Abstract
The macular pigments (MP) absorb light in the blue–green region of the visible spectrum and comprise two carotenoids, lutein
and zeaxanthin. In humans the concentration of MP varies widely across the normal population. There are two (not mutually
exclusive) proposed roles for MP: to improve visual function and to act as an antioxidant and protect the macula from damage by
oxidative stress. In this article we review the origin, spectral characteristics and ocular distribution of MP and also discuss the effect
MP has on central visual function and the techniques available for measurement of MP optical density in vivo. Finally, we review
the evidence for both proposed physiological roles of MP. Considering the first of these, we conclude that although MP might
improve visual function in theory, to date there is no firm evidence that higher levels of MP are correlated with enhanced measures
of visual performance. There is a growing body of evidence that has highlighted associations between macular disease and low levels
of MP, most particularly with age-related macular degeneration (AMD) and with risk factors for AMD. However, all findings to
date are associative only and there is no direct evidence for high MP levels conferring a protective effect. Increased dietary intake of
MP gives rise to increased levels of serum and retinal MP. This, taken together with the associative evidence of low MP levels in
disease, indicates that a potential, and perhaps serendipitous, therapeutic strategy for macular disease exists. We conclude, however,
that the potential protective properties of MP will only be fully evaluated by undertaking longitudinal studies that follow initially
healthy participants through to the development of macular disease.
r 2004 Elsevier Ltd. All rights reserved.
Contents
1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
534
2.
What are the MPs? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
534
3.
Where do the MPs come from? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
535
4.
Where are the MPs located? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
536
5.
Spectral properties of the MP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
537
6.
The effect of macular pigmentation on visual responses . . . . . . . . . . . . . . . . . . . . . . . . . .
539
7.
Measuring the macular pigmentation in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1. Psychophysical techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2. Objective measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
540
540
542
8.
Factors affecting MPOD within normal populations .
8.1. Comparison between eyes . . . . . . . . . . .
8.2. Twin studies and genetics . . . . . . . . . . .
8.3. Gender differences . . . . . . . . . . . . . . .
8.4. Age . . . . . . . . . . . . . . . . . . . . . . .
544
544
545
545
546
*Corresponding author. Tel.: +44-17784-443520.
E-mail address: [email protected] (A.B. Morland).
1350-9462/$ - see front matter r 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.preteyeres.2004.05.004
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ARTICLE IN PRESS
N.P. Davies, A.B. Morland / Progress in Retinal and Eye Research 23 (2004) 533–559
534
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10. Dietary supplement of macular pigmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
551
11. Putative roles for MP . . . . . . . . . .
11.1. Role in improving visual function .
11.1.1. Chromatic aberration . . .
11.1.2. Visibility . . . . . . . . .
11.2. MPs as antioxidants . . . . . . . .
8.5.
8.6.
9.
8.4.1. Children and young adults . . . . .
8.4.2. Prenatal, neonatal and infants . . .
MPOD in different populations . . . . . .
Other factors influencing MPOD in normal
8.6.1. Tobacco smoking . . . . . . . . .
8.6.2. Iris colour . . . . . . . . . . . . .
8.6.3. Lens density . . . . . . . . . . . .
8.6.4. Obesity . . . . . . . . . . . . . . .
Macular pigmentation in disease .
9.1. AMD . . . . . . . . . . . .
9.2. Diabetes . . . . . . . . . .
9.3. Other conditions . . . . . .
9.3.1. Albinism . . . . . .
9.3.2. Choroideraemia . .
9.3.3. Retinitis pigmentosa
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12. Outstanding issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.1. The importance of measuring the distribution or peak MPOD . . . .
12.2. How should macular pigmentation be measured? . . . . . . . . . . .
12.3. Can macular pigmentation be modulated to serve a protective role? .
12.4. Correlation and causality . . . . . . . . . . . . . . . . . . . . . . .
12.5. Mechanisms of deposition of the MPs . . . . . . . . . . . . . . . .
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13. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
556
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
556
The first documentation of a yellow colour in the
centre of the retina was made by Buzzi (1782) and a few
years later Soemmering (1799) was of the opinion that
the yellow spot represented a central retinal hole.
Maxwell (1856) made the observation that large,
spatially uniform coloured stimuli were often perceived
to have a central dark region. It was also noted that the
spot was particularly marked when stimuli included
short-wavelength (SW) light (Maxwell, 1856). Schultze
(1866) hypothesized that such SW absorption may
reduce the consequences of chromatic aberration and
may also play a protective role.
Although a phenomenon consistent with macular
pigmentation had been documented behaviourally,
Gullstrand felt that the yellow colour was a post
mortem change and not present in vivo (Gullstrand,
1907). In 1945, Wald showed that the absorption
spectrum of the yellow pigment was characteristic of a
carotenoid (Wald, 1945). Since Wald’s study, there has
been an explosion in efforts to chemically identify the
macular pigment(s) (MP), develop methodology to
measure and characterize MP in vivo, and understand
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1. Introduction
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the physiological role of MP. More recently, great
interest has developed in the potential role that MP may
play in macular disease. In this article, we will
concentrate on the work undertaken in the last 50 years
that has shed light on these questions. Despite the large
body of work, significant issues remain unresolved that
may bring into question the validity of the two current
working hypotheses for the role of MP: firstly, to
improve visual function, and secondly, to protect the
macula from oxidative stress.
2. What are the MPs?
Wald (1945) identified MP as belonging to the
Xanthophyll family. The first separation of the carotenoids from the macula was made much later by Bone
et al. (1985) using high-performance liquid chromatography (HPLC). Chromatograms obtained in this study
consistently showed the presence of two components.
Bone et al. (1985) labelled these components with the
neutral terms MP1 and MP2 and used experimental
procedures to determine the chemical identity of these
constituents. Firstly, the chromatograms were to all
ARTICLE IN PRESS
N.P. Davies, A.B. Morland / Progress in Retinal and Eye Research 23 (2004) 533–559
535
H3C
H3C
CH3
CH3
CH3
HO
OH
CH3
H3C
CH3
CH3
CH3
Lutein
H3C
H3C
CH3
CH3
CH3
HO
OH
CH3
CH3
H3C
CH3
CH3
Zeaxanthin
Fig. 1. The chemical structures of lutein and zeaxanthin, the principal constituents of the macular pigmentation.
intents identical to chromatograms obtained from lutein
(L) and zeaxanthin (Z) standards. Secondly, the
absorbance spectra of MP1 and MP2 could also be
identified with those of L and Z. Thirdly, evidence of the
identification of MP1 with lutein and MP2 with
zeaxanthin was made based on the subtle difference in
chemical structures of L and Z. L has an allylic hydroxyl
group which can be converted to a methyl ester. Z,
however, does not undergo the same reaction. Performing this chemical reaction on MP1 and L standard,
followed by HPLC, gave two new compounds with
identical elution times on the HPLC column. The same
procedure was conducted on MP2 and the Z standard.
The HPLC results showed a single compound only, for
both MP2 and Z, and this peak was identical to that of
untreated zeaxanthin. The feature that the MP comprised two components was later confirmed by Handelmann et al. (1988). It is very important to note,
therefore, that the pigmentation of the macula does
not arise from the presence of a single carotenoid, but
rather the presence of two major carotenoid constituents. The two carotenoids (members of a 40 strong
family of carotenoids) are depicted in chemical form in
Fig. 1.
3. Where do the MPs come from?
L and Z are not synthesized within the body and
therefore the MPs have to be provided by dietary intake.
Wald (1945) originally identified the MPs as having the
spectral properties of a xanthophyll, which originate in
the leaves of green plants. More recent work has allowed
the relative concentrations of the MPs in foodstuffs to
be assessed more thoroughly. Khachik et al. (1992) used
HPLC to assess levels of carotenoids in fruits and
vegetables. These studies had the disadvantage of not
separating the individual concentrations of L and Z. As
L and Z are found in different distributions in the retina,
Sommerburg et al. (1998) used HPLC to analyse the
contents of different fruits and vegetables for each MP
constituent. Their findings are presented in Table 1,
reproduced from the original paper. The results agreed
with those from a previous paper collating data from
1971 to 1991 (Mangels et al., 1993), but quantify the
distribution of L and Z in the different foodstuffs.
An important development in recent years has been
the analysis of carotenoid concentration in the human
plasma in vivo (Khachik et al., 2002) and how
concentration is influenced by diet. Primates fed a
carotenoid-free diet had no detectable yellow pigmentation in the macula (Malinow et al., 1980) and levels of
MP in humans (measured using heterochromatic flicker
photometry) can be raised by dietary supplementation
(Bone et al., 2003; Hammond et al., 1997a; Landrum
et al., 1997). The underlying mechanisms of incorporation of MP into retinal tissues are poorly understood.
The increase in optical density of MP with oral
supplementation is slow, rising steadily and reaching a
plateau after 140 or more days of supplements containing 30 mg of L (Landrum et al., 1997). Following
cessation, supplement levels fall at a slower rate
and have been shown to remain raised for a period of
at least 6 months in some individuals (Hammond et al.,
1997a).
It is also now possible to buy lutein and zeaxanthin
concentrated in tablet form, either alone or in combination with other health products. A simple Internet
search reveals a large number of retailers offering such
preparations.
ARTICLE IN PRESS
536
N.P. Davies, A.B. Morland / Progress in Retinal and Eye Research 23 (2004) 533–559
Table 1
Macular carotenoid content of fruits and vegetables given in mol%
Vegetable/fruit
Lutein and
zeaxanthin
Lutein
Zeaxanthin
Egg yolk
Maize (corn)
Kiwi
Red seedless grapes
Zucchini squash
Pumpkin
Spinach
Orange pepper
Yellow squash
Cucumber
Pea
Green pepper
Red grape
Butternut squash
Orange juice
Honeydew
Celery (stalks, leaves)
Green grapes
Brussels sprouts
Scallions
Green beans
Orange
Broccoli
Apple (red delicious)
Mango
Green lettuce
Tomato juice
Peach
Yellow pepper
Nectarine
Red pepper
Tomato (fruit)
Carrots
Cantaloupe
Dried apricots
Green kidney beans
89
86
54
53
52
49
47
45
44
42
41
39
37
37
35
35
34
31
29
29
25
22
22
20
18
15
13
13
12
11
7
6
2
1
1
0
54
60
54
43
47
49
47
8
44
38
41
36
33
37
15
17
32
25
27
27
22
7
22
19
2
15
11
5
12
6
7
6
2
1
1
0
35
25
0
10
5
0
0
37
0
4
0
3
4
0
20
18
2
7
2
3
3
15
0
1
16
0
2
8
0
6
0
0
0
0
0
0
4. Where are the MPs located?
The MPs, as the name suggests, are most dense within
approximately the central 7 mm2 of the human retina.
To the first approximation, the overall distribution of
the pigments peaks in the fovea and gradually decreases
with increasing eccentricity, a feature that can be readily
observed in fundus photographs. The nature of the
spatial distribution of the human MPs has also been the
focus of quantitative evaluation. Psychophysical techniques have revealed the spatial profile of the MPs in
human in vivo (Hammond et al., 1997a,c; Moreland and
Bhatt, 1984). It has also been noted that the decrease in
MP density with eccentricity is not necessarily monotonic (Moreland and Bhatt, 1984; Robson et al., 2003).
This feature could arise from the two principal chemical
constituents varying in concentration differently across
the retina (Bone et al., 1988). It also seems clear that the
spatial extent of the MP distribution varies between
subjects (Hammond et al., 1997c; Moreland and Bhatt,
1984; Robson et al., 2003) and the extent also appears to
be influenced by age (Chang et al., 2002). The
psychophysical evaluations of the spatial profile of MP
density are painstaking and lengthy measurements.
Also, the resolution of such measurements is limited to
the size of the visual stimulus that is presented (most
often circa 1 2).
Imaging techniques, which can obtain information
over a larger area of the retina, overcome many of the
shortcomings of behavioural assessments of the spatial
distribution of MP in the human retina. The pioneers of
this technique (Kilbride et al., 1989) revealed that their
data were best fit by a Gaussian function of eccentricity
centred on the fovea, measurements being made at every
0.1 . More recent use of imaging techniques has allowed
even greater spatial resolution to be achieved, but not all
have reproduced Kilbride et al.’s finding that the
distribution is Gaussian (Robson et al., 2003). Robson
et al. (2003) also highlighted an interesting dissociation,
where the overall amount of macular pigmentation does
not correlate with the peak density. It remains to be seen
whether the peak density or total amount represents the
most prudent measurement to quantify macular pigmentation if its enhancement as a treatment for disease
is to be assessed.
In addition to the work undertaken on the overall
topography of macular pigmentation in humans, other
studies on non-human primates have been able to reveal
the distribution of the pigments in the retinal layers.
Snodderly et al. (1984b) used photographic and microspectrophotometric techniques to evaluate the distribution of MP in the layers of the primate retina. A
companion paper used microdensitometry to investigate
the spatial distribution of the MP across the retina
(Snodderly et al., 1984a). Microphotographs of prepared sections of foveal tissue obtained from Macaca
fascicularis, M. mulatta and M. nemestrina were taken
using a 460 nm spectral primary and also with a primary
of 525 nm. The bands of high absorption of the SW light
were seen in the photoreceptor axon layer and in the
inner plexiform layer. To identify whether these
absorption bands in the different retinal layers contained the same or different pigments, the absorption
spectrum of single retinal layers was measured. The
measuring beam had a cross-sectional diameter of 10 mm
and absorbance values at 5 nm intervals from 400 to
500 nm and at 10 nm intervals from 500 to 600 nm were
taken. Using a computational technique and a series of
template spectra derived by taking the difference in
absorption between two nearby locations in the specimen under study, they were able to show that the
majority of yellow pigments in the photoreceptor axon
layer and the inner plexiform layer of the fovea were
MPs, with a peak absorbance of 460 nm. They also
identified two other SW filters, with peaks of absorbance
ARTICLE IN PRESS
N.P. Davies, A.B. Morland / Progress in Retinal and Eye Research 23 (2004) 533–559
at 410 and 435 nm, which were named P410 and P435,
respectively. Spectra taken at increasing eccentricity
from the centre of the fovea showed a rapid decrease in
the density of MP in the photoreceptor axons, to reach
the levels found in the other retinal layers by 400 mm
eccentricity.
The P410 filter showed an increase with eccentricity,
whilst the P435 filter showed a decrease with eccentricity. The P410 filter levels in the receptor axon layer
decreased with increasing eccentricity. In the inner
retinal layer, the levels of P410 were higher and the
net effect of this overall was to lead to a small but steady
increase in the level of P410 with eccentricity. The P435
levels showed a steady increase in the receptor layer, but
always existed in low levels in the inner layers. The
increasing volume of the inner layers with eccentricity
lead to an overall reduction in P435 density with
eccentricity. The P410 and P435 pigments have spectra
that can be identified with the haemoproteins reduced
cytochrome C and oxidized haemoglobin, respectively.
In the second paper (Snodderly et al., 1984a), the
spatial distribution of MP was studied using twowavelength microdensitometry. Foveal tissue from
primates was prepared and scanning microdensitometry
was performed at two wavelengths, 460 and 525 nm. The
spatial profile of difference in absorption between the
two wavelengths was overlaid on images of the retinal
layers traced from microphotographs. This method
allows the peaks of absorbance in the different fibre
layers of the retina to be seen. Interestingly, the relative
amounts of the MP in the receptor axons and the inner
plexiform layer varied considerably between specimens.
In some specimens the peak density of pigment in the
receptor axons exceeded that of the inner plexiform
layer and the IPL density decreased rapidly with
increasing eccentricity. In other specimens, the pigment
density in the inner plexiform layers declined less rapidly
and hence exceeded the receptor axon pigment density
at eccentric locations.
The relative distribution of L and Z across the macula
has also been investigated (Bone et al., 1988). In adult
retinae Z is clearly dominant in the centre of the fovea,
with the amount reducing with eccentricity. There is a
concomitant rise in the relative level of L with increasing
eccentricity. The L:Z ratio changes from 1:2.4 centrally
to 2:1 peripherally. The reason for this change in
distribution is unclear, although the authors offered a
tentative explanation. A plot of L:Z ratio against the
rod:cone ratio obtained in another study from one
individual showed a linear relationship. The suggestion
was made that Z may be associated with cones and L
with rods. This is, however, an inductive step from the
two data sets and the direct association of different
carotenoids with different receptor types remains
unconfirmed. However, since that study, both L and Z
have been shown to be associated with rod outer
537
segments (ROS). Sommerburg et al. (1999) demonstrated that about 25% of the total retinal carotenoid is
found in the ROS. Rapp et al. (2000) has also measured
the L and Z concentrations in ROS following the
argument that receptor outer segments are the sites most
prone to damage from oxidative stress. The results again
showed the presence of both L and Z in the membranes
of ROS, representing approximately 10–15% of the
total retinal amount of the MP. The ratio L:Z also
varied with eccentricity, increasing from 1.8 in the
perifovea to 2.68 in the peripheral sample.
Although the carotenoids L and Z are concentrated in
the macula, they are also present in non-central retina
and in other ocular structures. Several studies to date
have investigated the presence of the MP carotenoids in
ocular tissues. Bernstein et al. (2001) used human donor
eyes to identify and quantify carotenoids in the ocular
tissues, using HPLC. The aim of the study was to
characterize the complete carotenoid profile of the eye;
here we present the results for the macular carotenoids
only. Globes were obtained within 24 h of death and
corneas harvested for transplantation. Corneas rejected
for transplantation on the basis of serum antigenicity
were used in the study. The remaining ocular tissues
were dissected and separated to give irides, ciliary body,
lens, vitreous, retina, and RPE/choroid. The retinae
were divided into macular retina (5 mm trephine), four
samples of mid-peripheral retina (superior, inferior,
nasal and temporal) and the remaining peripheral retina.
The RPE/choroid samples were peeled from the retinal
samples after trephination. Both individual and pooled
tissue samples were prepared, the latter to aid detection
of low levels.
The levels of L and Z obtained from pooled extracts
and individual ocular tissues are given in Table 2. The
results clearly show that although L and Z are
concentrated in the macular retina, they are also present
in the majority of ocular tissues, with the exception of
the vitreous. Trace levels only were found in cornea and
sclera.
5. Spectral properties of the MP
The spectral absorption properties of macular pigmentation have been subject to study for over 50 years.
The studies can be divided into in vivo and in vitro
measurements. The early in vivo measurements were
derived from spectral sensitivity measurements (Brown
and Wald, 1963; Stiles, 1953; Wald, 1949), colour
matching (Ruddock, 1963), and visualization of Haidinger’s brushes (de Vries et al., 1953; Naylor and
Stanworth, 1954). The spectra derived in some of these
studies were brought together by Wyszeski and Stiles
(1982) in order to generate a ‘preferred mean curve’. The
mean curve was a weighted mean of the spectra derived
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Table 2
Lutein and zeaxanthin levels in human ocular tissues
Ocular region
Eyes examined
Area (mm2)
Lutein (ng per tissue 7SD)
Zeaxanthin (ng per tissue 7SD)
L/Z ratio
Macular retina
Peripheral retina
Superior retina
Inferior retina
Nasal retina
Temporal retina
RPE/choroid
Superior RPE/choroid
Inferior RPE/choroid
Submacular RPE/choroid
Ciliary body
Iris
Lens
Cornea
Sclera
Vitreous
14
19
78
78
7
7
17
78
78
25
20
21
18
3
5
3
20
E1000
20
20
20
20
Whole
20
20
20
Whole
Whole
Whole
Whole
20
0.5 ml
13.9873.58
64.18730.10
1.6870.88
1.4670.71
1.7671.01
1.4270.90
11.5875.99
0.6370.26
0.5370.26
0.7770.50
12.7277.90
4.0371.98
1.6671.09
Trace
Trace
Not detected
19.0674.5
34.11716.83
0.8070.62
0.6370.26
0.8170.53
0.6570.42
5.8974.13
0.1970.09
0.1670.09
0.3270.20
5.9873.50
1.5470.98
1.4371.20
Trace
Trace
Not detected
0.7
1.9
2.1
2.3
2.2
2.2
2.0
3.3
3.3
2.4
2.1
2.7
1.2
—
—
—
Macular Pigment Spectra
0.6
0.5
Optical Density (log units)
from the studies, with weights being proportional to the
ease of the measurements. This curve has been used
extensively since its publication. However, there have
been more recent in vivo measurements that indicate
departures from the adopted standard. Pease et al.
(1987) derived a spectrum psychophysically and reported the spectrum of the MPs having considerable
absorption beyond 535 nm. The extent of the longwavelength (LW) absorption was, however, not nearly
as great when Sharpe et al. (1998) derived a spectrum
from sensitivity measurements of isolated cone mechanisms (some MP spectra are compared in Fig. 2).
The chemical identification of the MPs as L and Z has
allowed the absorption spectra of these constituents to
be determined. In general, the constituents absorb most
strongly in the blue–green region of the spectrum (circa
460 nm). Significant absorption in the pigments also
occurs at wavelengths beyond the lower limit of visibility
(o380 nm). The chemical constituents do not appear to
absorb light strongly beyond wavelengths of 530 nm. It
should be noted, however, that the chemical environment of L and Z might play some role in modifying the
absorption characteristics of macular pigmentation. To
address this issue, Bone et al. (1992) mixed L and Z in
appropriate quantities and chemically mimicked the
conditions under which L and Z would be found in the
eye. The resultant spectrum of the L and Z mixture,
modified by the presence of a bilipid membrane, showed
very good agreement with psychophysical measurements
made in the same study. It should also be noted that a
more recent psychophysical investigation (Sharpe et al.,
1998) indicated that the in vitro spectrum derived by
Bone et al. (1992) explained better the spectral data
derived by Sharpe et al.
A noteworthy feature of the spectrum for the L and Z
mixture derived by Bone et al. (1992) is the light
W&S
Ruddock
Bone
Pease
0.4
0.3
0.2
0.1
0
-0.1
380
430
480
wavelength (nm)
530
580
Fig. 2. MP density spectra. Data are given for studies by Ruddock
(1963), Bone et al. (1992) and Pease et al. (1987). The thick solid line
labelled W&S is the spectrum derived from different studies
summarized by Wyszeski and Stiles (1982).
absorption at wavelengths beyond 530 nm. This feature
differentiates the spectrum from those put forward by
Wyszecki and Sitles (1982), which indicates insignificant
absorption beyond 530 nm (Fig. 2).
Is it now time to advocate a standard spectrum? It
might be argued that Bone et al.’s (1992) spectrum
should be adopted as it represents the most precise
measurement of the absorption characteristics of a likely
combination of L and Z. It is worth noting that no
single spectral template is likely to be appropriate for
each individual. The principal reason for this is that the
relative concentrations of L and Z, pigments that have
different absorption spectra, will vary from one observer
to another (perhaps due to diet) and also with retinal
location in each individual (Bone, 1976). It should also
be noted that the oxidation products of L and Z may
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exist in significant concentration in the macula and their
presence may perturb further the spectral absorption
due to the overall macular pigmentation. It is feasible,
therefore, for two subjects with the same absorption of
light at 460 nm to have differing absorption of light at
other wavelengths because of the different proportions
of the constituents of MP. As Sharpe et al. (1998) point
out, it may be impossible to reach a consensus about the
spectrum of the MPs. However, if one template is
required, it is the view of the authors that Bone et al.
(1992) provide the best spectrum at present.
6. The effect of macular pigmentation on visual responses
The MPs, as explained earlier, exhibit selective
absorption of light in the visible spectrum. The majority
of this absorption occurs before light is incident on the
photoreceptors. The effect of the pigments therefore is
to change a known spectral distribution of light incident
on the cornea, the external stimulus, to an unknown
spectral distribution at the macular photoreceptors, the
internal stimulus. It should also be noted that other
spectrally selective absorptions occur to the external
stimulus, the most significant being that of the lens. In
changing the external stimulus, the MPs can have
profound effects on spectral responses that are specified
in terms of the physical properties of the external light
stimulus. In fact, this very effect is relied upon to
estimate the density of the MP with psychophysical
techniques in vivo.
Because of inter-observer variations in macular
pigment optical density (MPOD) (Bone and Sparrock,
1971), visual responses can be profoundly different
between subjects, particularly at wavelengths where the
MPs absorb most light in the blue–green region of the
visible spectrum. If underlying mechanisms of human
vision need to be quantitatively evaluated, the variability
in visual responses associated with macular pigmentation needs to be minimized or corrected for. In other
words, an estimate of the spectral distribution of light
incident on the photoreceptors is required if the function
of the photoreceptors and mechanisms beyond them
need to be probed quantitatively.
In order to illustrate how spectral responses can be
modified by inter-observer variations in macular pigmentation, we briefly present a study of the LW sensitive
cone spectral response for 10 deuteranopic observers.
Such measurements are essential for generating spectral
primary functions for the specification and reproduction
of coloured stimuli and have been documented by a
number of groups with different methods (Smith and
Pokorny, 1972, 1975; Vos and Walraven, 1971; Walraven, 1974). Here we measure the spectral responses using
a colour-matching method devised by Maxwell and used
in a study by Alpern and Pugh (1977), who fully
539
elaborate on the method used. The data derived from
the colour-matching procedure can be plotted as a
spectral response for each subject (Fig. 3A).
The responses have been normalized to the mean of
sensitivities acquired at wavelengths beyond 600 nm,
where the MPs do not absorb light. The variance in the
responses over the shorter wavelengths is suggestive of
variations in the density of macular pigmentation
between subjects. A model of the effect of the MP on
an ideal spectral response is shown in Fig. 3B. The ideal
response displays the highest sensitivity over shorter
wavelengths, and the effect of screening by macular
pigmentation of densities 0.4 and 0.8 is shown by the
systematic reduction of sensitivity over shorter wavelengths in the other two responses. Qualitatively, the
model reflects the observed variations across subjects in
Fig. 3A. The model can therefore be adapted to
determine quantitatively how much screening due to
macular pigmentation is required to align an ideal
spectral response to the response of an individual
subject. This is equivalent to calculating the MP density
for each subject.
A useful procedure is to then remove the effects of the
estimated macular pigmentation from each subject’s
spectral response to determine what remaining variance
exists between subjects. This procedure results in the
plot shown in Fig. 3C, which indicates that the variance
over the shorter wavelengths is reduced considerably
once the effects of inter-subject variability in macular
pigmentation are accounted for. Although the correction of the spectra for variations in macular pigmentation provides a self-consistent way of reducing intersubject variability over shorter wavelengths, it is also
desirable to determine whether the magnitude of the
corrections made to the spectral data correlate with a
more direct estimate of macular pigmentation. In Fig. 4,
we show how the estimate of macular pigmentation
evaluated from the spectral responses relates to another
measure that we derived from Ruddock’s (1963; also see
below) colour-matching method. The correlation between the estimate and measurement is very high and
allows the modelling to be verified.
The data presented are shown as an illustration of
how much inter-subject variation in visual response can
be caused by macular pigmentation. However, the
illustration we have used only indicates how macular
pigmentation causes changes a spectral response when
data were obtained with a stimulus that extended over
the central 1.3 . Macular pigmentation is known to
show a decrease in density with retinal eccentricity and
can therefore cause problems if spectral responses need
to be compared at different retinal locations within a
subject.
A consequence of spatial variations in pigment
density is that the luminous efficiency function, Vl,
will be different at different retinal locations in an
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540
deuteranopes'
uncorrected responses
deuteranopes'
corrected responses
model
1.0
log(relative sensitivity)
0.5
0.0
-0.5
-1.0
-1.5
450
500
(A)
550
600
650
700 450
wavelength (nm)
500
550
600
650
wavelength (nm)
(B)
700 450
(C)
500
550
600
650
700
wavelength (nm)
Fig. 3. (A) Spectral responses reflecting the sensitivity of the long-wavelength cone mechanism obtained for 10 D. Responses were normalized to the
mean sensitivity obtained at wavelengths X600 nm. (B) An ideal spectral response (the uppermost curve) and responses derived from that curve by
modelling the effects of light absorption in the (MPs) due to optical densities of 0.4 (middle curve) and 0.8 (lower curve). (C) Spectral responses of
10 D that have been modified in line with light absorption in the MPs (see text for details).
fitted MP optical density
0.8
0.6
0.4
0.2
0.0
-0.2
0.0
0.2
0.4
0.6
0.8
estimated MP optical density
Fig. 4. MP density derived from the spectral responses shown in Fig. 3
(fitted MP) plotted as a function of the MP density (estimated MP)
derived from the colour-matching method described by Ruddock
(1963). Note that the estimated MP density was measured at 460 nm
and that the fitted MP density was derived from measurements made at
500 nm and above. The line of best fit (solid line) is described by fitted
MP=0.988 estimated MP+0.006 (R2=0.872). The gradient is
essentially unity, which indicates that the estimate of MPOD at
460 nm is an excellent predictor of variations in the spectral responses
caused by macular pigmentation for wavelengths greater than 500 nm.
The spectral absorption characteristics of the MP used in the
modelling were those derived by Ruddock (1963).
individual. The knock-on effect of this variation is that
specification of equiluminant stimuli has to vary with
retinal eccentricity in an individual. This causes pro-
blems for investigators who are interested in determining the properties of SW sensitive mechanisms over an
extended region of retina. Such investigations are prone
to having spatially varying luminance signals in stimuli,
which do not allow the action of SW chromatic
mechanisms to be disambiguated from responses of
achromatic mechanisms. At first, this may appear only
to be of interest to the few psychophysicists who
investigate retinal processing of colour in humans, but
objective measurements of clinical relevance can also
rely on isolating chromatic from achromatic mechanisms. For instance, the blue-cone ERG measurement
needs to record a signal that principally reflects the
response of blue cones. Unfortunately, the MP absorbs
strongly at wavelengths that would be best used to
stimulate blue cones. Methods have been developed that
can overcome these issues. For instance, the use of very
bright LW background lights helps suppress luminance
detection mechanisms, whilst leaving the sensitivity of
blue-cone mechanisms little changed (Chiti et al., 2003).
7. Measuring the macular pigmentation in vivo
7.1. Psychophysical techniques
Because the MP has profound effects on human
perception of light, it has been relatively straightforward
to use psychophysical techniques to assess MPOD in
individuals. In fact, psychophysical measurements preceded the chemical identification of the MPs by many
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decades. We will describe the contemporary psychophysical techniques that are used to determine the properties
of the MPs and provide a critique of each. We will not
describe methods that involve visualization of Haidinger’s brushes, but note here that early evaluations of
MPOD did implement such a technique (de Vries et al.,
1953; Naylor and Stanworth, 1954).
As we have already outlined, the MPs modify the
spectral content of a light stimulus incident on the
macular photoreceptors. In order to assess the extent to
which the spectral content of the stimulus has been
modified by the MPs, psychophysicists have frequently
compared foveal with extrafoveal responses. In taking
this approach, two conditions need to be met: (1)
different concentrations of MPs need to be present at
the foveal and extrafoveal locations and (2) the response
measured is influenced only by the difference in MP
concentration at the two locations. As we will describe,
the first condition is generally met, but the extent to
which the second is met varies considerably between
different techniques. It could also be argued that the
second condition may never be met in full.
The human luminous efficiency curve, Vl, can be
derived from absolute threshold measurements, heterochromatic flicker photometry (HFP) or motion photometry. From quantitative evaluations of Vl, it was clear
that for foveal targets considerable inter-subject variability in sensitivity existed and was particularly pronounced for SWs (Stiles, 1953; Wald, 1949). These
measurements indicated that macular pigmentation
could be quantified if a comparison of the sensitivity
achieved in the fovea were compared with a response
obtained from the same observer at a more eccentric
retinal location. The difference between the two
responses as a function of wavelength provides an
estimate of the spectral absorption of the MPs.
The accuracy of the estimate would, in this case,
depend on at least three factors: (1) the photopigment
density in the cones at the two locations, (2) rod
intrusion to the response at extrafoveal locations and (3)
the relative numbers of each cone class at the two retinal
locations. The change in photopigment density with
retinal location is a very difficult issue to overcome.
Moreover, photopigment density may also undergo
pathological modification in disease, which in turn
could affect estimates of MPOD in patients. The second
effect of rod intrusion can be reduced by selecting an
extrafoveal location that is not too eccentric, and by
using high-luminance stimuli.
The remaining problem of relative numbers of cones
varying in different locations is overcome when sensitivity is mediated by only one detector (cone class). Stiles
(1949) used large, bright background stimuli to selectively reduce the sensitivity of some cone mechanisms
over others, and thereby render the detection threshold
dependent on one cone mechanism alone. By using such
541
a technique, Pease et al. (1987) successfully derived
macular pigmentation estimates for 27 observers and
also derived spectra for the MPs in 12. This method
offers, therefore, an advantage over measuring overall
sensitivity because of the fewer assumptions involved.
The most commonly adopted method of HFP can also
be adapted to suppress the sensitivity of the S-cone
mechanism (Hammond et al., 1998). Although sensitivity still remains a product of the M and L cone
mechanisms under these conditions, it is particularly
desirable to exclude the spatially varying effects of the Scones on sensitivity measures (Hammond et al., 1998). It
should be noted that there has been some recent debate
concerning the effects of isolating different cone
mechanisms on the estimates of MP density spectra
(Sharpe et al., 1998).
An elegant method to estimate the spectral absorption
due to macular pigmentation was pioneered by Ruddock (1963, 1965) and later developed by Moreland (e.g.
Moreland and Kerr, 1978). The colour-matching protocol requires that the observer adjust the energy of
three monochromatic light stimuli to match a single
monochromatic test stimulus. The monochromatic test
stimulus receives no spectral change due to the absorption of MPs. However, the triplet of matching stimuli
receives differential absorption by the MPs. For
instance, the MPs will absorb an SW more than an
LW stimulus. The effect of the differential absorption
results in more SW light being needed for a colour
match measured at the fovea than for one established at
a more eccentric location. Colour matches can be
performed with the choice of different wavelengths
and a full spectral characterization of the MPs can
therefore be derived (see Ruddock, 1963 and Fig. 2).
The quantification of the colour match readily reveals
the magnitude of the MP density (Ruddock, 1963).
Equipment and efficient methods have been established
to measure MP density with colour matching (Moreland, 1980). Moreover, selection of matching stimuli
wavelengths that are equally absorbed by the MPs allow
the effects of macular pigmentation on colour matches
to be negated (Moreland and Kerr, 1979; Ruddock,
1963). The so-called Moreland match uses such stimuli
and is a useful tool to probe the function of the SW
sensitive cones and therefore deficiencies of this cone
mechanism (Moreland and Kerr, 1979). One problem
with all psychophysical procedures is that a ‘MP-free’
measurement has to be made at an eccentric stimulus
location, which is demanding for the observer. Some
studies (Moreland and Bhatt, 1984; Moreland et al.,
2003) have used large annular targets, which are less
demanding on the observer, and standard errors of
matches are reduced for such a stimulus arrangement
(Morland, 1992).
The measurements of MP density with colour
matching are not subject to changes in the relative
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number of cones with retinal location, but in common
with sensitivity measurements, rod intrusion could play
a role in modifying matches made at eccentric locations.
To overcome rod intrusion, bright stimuli can be used.
Again, variation in photopigment density with retinal
location is the most difficult obstacle to overcome.
However, it is possible to model what effect it might
have on results. We have undertaken such modelling
(Davies and Morland, 2002) and find that for photopigment density variations that might be expected in the
healthy retina, the change in MP density estimated from
colour matching varies little for the wavelengths used in
our study.
Up to now, we have focussed on how sensitivity
measurements and colour matching have been used to
obtain a detailed picture of the spectral characteristics of
the MPs. There is perhaps a far greater demand now for
obtaining a quick estimate of peak MP density for a
large number of individuals, because of the putative role
that MPs may play in protecting the retina. This
demand can be met by employing both of the methods
we have outlined. A selection of just two wavelengths at
which sensitivity is measured at two retinal locations can
provide an estimate of macular pigmentation. For
example, measuring spectral sensitivity at a LW, say
600 nm, where no absorption due to macular pigmentation occurs, provides a useful point to normalize the
foveal and parafoveal sensitivities. The other measurement of sensitivity should be made at a wavelength
where MPs absorb strongly, for example 460 nm. In this
case, only a few measurements are necessary, a feature
that makes the method reasonably quick to implement
and applicable to large numbers of observers. Many
groups have used such techniques extensively in their
studies of the factors that influence macular pigmentation (see Section 8). Similarly, just two colour matches
using appropriately selected wavelengths can provide an
estimate of peak MPOD with reasonable speed. It is
interesting to note that colour matching has not been
used to survey large samples.
Many variants of psychophysical techniques have
been employed to assess MPOD. We now consider
which techniques represent the most promising approaches and how they can be optimized to allow
comparison across studies. The ideal technique needs to
conform to three demands: (1) it needs to be readily
implemented in a reduced form so that data from a large
group of observers can be readily obtained; (2) it needs
to reduce the effects of confounding variables as much
as possible; and (3) in an extended form, it should
provide a viable method for determining the absorption
spectrum of the MPs in vivo. The third demand could
perhaps be met by specialized laboratories where the
extended form of the test may be possible. The first two
demands should be met and can be done by implementing colour-matching methods or by determining the
sensitivity mediated by a single-cone mechanism. Both
techniques offer the same sort of advantages, so it would
not seem appropriate to recommend one over the other.
Recommendations can be made to increase the
likelihood that the data derived from both techniques
could be compared. Standardizing the wavelength,
locations and size of target stimuli would allow
psychophysical measurements to be compared with the
fewest assumptions. These three factors are of undeniable importance if a comparison of MP density across
different studies is required. It may be useful to
implement a choice of these parameters, which has
some backward compatibility with studies that have
already been performed on large groups of subjects.
Unfortunately, many previous studies have used an
eccentric reference stimulus that is presented at 4 . This
choice has the consequence of introducing errors in
estimates of peak MPOD that are due to intersubject
variations in the spatial profile of MP concentrations
(Moreland and Bhatt, 1984; Robson et al., 2003).
Recent work indicates that a choice of 7 for the
location of the eccentric target is more appropriate
(Robson et al., 2003). The other stimulus features of
previous studies have fewer problems associated with
them. It would appear reasonable to recommend the
following choices: (1) a central stimulus of 1 diameter,
(2) wavelengths of 460 and 600 nm, (3) an eccentric
reference location of 7 . To underscore the importance
of size of the central stimulus, it is worth quoting the
change in estimated MPOD that Bone et al. (1997)
reported in an observer when targets with different
diameters were used: for targets of 0.25 , 0.5 , 1.0 ,
1.6 , the ‘peak’ MPOD was estimated at 0.92, 0.80, 0.75
and 0.57. The effect of spatial configuration of stimuli
on estimates of MPOD can also be readily seen in the
data presented in Fig. 5, where two stimulus types are
compared. Also of note is the relatively high mean
MPOD of 0.77 recorded by Pease et al. (1984) in their
study, which used a 40 min target.
Although the psychophysical techniques we have
described can be optimized for estimating MPOD, there
is one problem that may be insurmountable and of some
considerable relevance if measurements need to be made
on elderly observers or those with disease. This problem
concerns unstable fixation and eye movements, which
result in the experimenter not knowing exactly the
retinal locus of the stimulus (Abadi and Cox, 1992).
Objective measures that image the retina have the
distinct advantage of not being subject to such
uncertainties and will be reviewed next.
7.2. Objective measures
It may well be apparent to the reader that the
psychophysical techniques leave a lot to be desired most
particularly because there are confounding variables and
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543
5
4
1.33deg
N
3
2
1
0
8
N
2deg
6
5deg
4
10deg
2
0
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
MPOD
Fig. 5. MPOD measured at 460 nm with colour matching (Ruddock, 1963). The upper panel shows the frequency distribution of MPOD for a group
of 13 normals measured with a 1.33 square bipartite field (see inset to the upper panel) presented foveally and 6 extra foveally. The lower panel
shows the MPOD frequency distribution for 30 normal subjects, but in this case the foveal bipartite field was a circle of 2 diameter and the extrafoveal filed was annular (see inset to the lower panel). The mean values of the distributions of 0.56 (upper panel) and 0.38 (lower panel) were
significantly different (independent t test, t ¼ 2:63; p ¼ 0:006 (two-tailed)).
no standards have been established to develop a
measurement protocol. An important issue is also
neglected by all psychophysical techniques, namely, that
a portion of the macular pigmentation may also exist
adjacent to the photoreceptors, rather than in front of
them. This portion of the macular pigmentation would
not be documented by psychophysical studies as it
would not affect the light incident on the photoreceptors. It is desirable, therefore, to have an objective
measurement that could be routinely applied in the
clinical setting, particularly if enhancing MP levels
becomes a therapeutic strategy. There are a few
objective techniques that have emerged and it is our
belief that some of these, perhaps with modification and
standardization, could become very useful tools in the
future, most particularly for the ophthalmologist.
A digitized image of the fundus (Fig. 6) shows
absorption of blue light that is characteristic of macular
pigmentation. The image shown was derived from a
digitized fundus image obtained from a Topcon 3 chip
CCD camera. The image was spatially filtered using a 2d
Gaussian and the log of each pixel for the blue and red
channels was taken and the difference calculated. The
subject underwent light adaptation at high luminance in
order to bleach the photopigments. Inspection of the
Fig. 6. An image of the fundus of one of the authors obtained by
taking the log difference of the blue and red channels of a digitized
fundus image taken with a Topcon camera. The data have also been
spatially filtered to increase the signal to noise.
image shows a characteristic, circularly symmetric
pattern of macular pigmentation. This demonstration
is entirely consistent with the images that Kilbride et al.
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(1989) obtained in their pioneering measurements of this
type. However, the image does not offer a precise way to
quantify MPOD because of the use of broad-band
detectors in standard fundus cameras. The introduction
of a narrow band filter in a fundus camera can yield
reliable correlates of MPOD and with this technique it
has been demonstrated that human albinos have little or
no MP (Abadi and Cox, 1992) and has also been used in
pediatric subjects (Bour et al., 2002).
Spectral fundus reflectometry represents a technique
that is capable of quantifying MPOD from fundus
images (Kilbride et al., 1989). However, there are some
issues that can detrimentally affect MPOD estimates
derived from this type of imaging technique. Firstly, it is
not possible to assert that the only spatially varying light
absorption at a SW is due to L and Z (Kilbride et al.,
1989). Both haemoglobin and melanin display spatially
varying light absorption, and unless a multiple regression analysis of spectral data is performed the relative
contributions of these absorptions cannot be accounted
for. It is also worth noting that such multiple regression
analysis is also dependent on an absorption spectrum of
the MPs (see Section 5 and also Bone et al., 1992) and
that this may vary between observers depending on the
relative amounts of the L and Z constituents. Secondly,
the measurements are not derived from a single pass of
light through the MP. To the first approximation, the
measurements are double pass, but this is undoubtedly a
poor approximation, because reflection of light does not
only occur at the RPE, but also the internal limiting
membrane (Berendschot et al., 2000). Scatter will also
play a role in perturbing estimates of MPOD, and this
will be a particularly significant problem in imaging
older eyes. Thirdly, autofluorescence of the lens and
lipofuscin can contribute to the light reflected from the
eye and hence cause problems with measurements of
MPOD.
Do these issues limit the accuracy of the spectral
fundus reflectometry measurements? The answer is yes,
but on the whole they are only likely to prevent an
absolute measurement of MPOD. Spectral fundus
reflectometry is, therefore, measuring a correlate of
MP. It has been found that this correlate is systematically lower than the value of MPOD determined by
psychophysical means (Delori et al., 2001; Kilbride et al.,
1989), but when a sophisticated model that accounts for
the light reflected from the inner limiting membrane is
used estimates of MPOD increase (Berendschot et al.,
2000) to levels that are similar to those derived from
psychophysics.
Scanning laser ophthalmoscopes (SLO) can also be
used to determine correlates of MPOD (Berendschot
et al., 2000). Images obtained at two wavelengths are
subtracted in a way similar to a standard image of the
fundus (as described earlier). Because the choice of
imaging wavelengths is restricted by the lasers em-
ployed, estimates of MPOD require scaling in line with
MP spectral absorption. However, the precision of these
measurements is higher than spectral fundus reflectometry measurements (Berendschot et al., 2000). The
accuracy of the SLO measurements may not however be
high, as systematically lower values of MPOD were
documented with this technique (Berendschot et al.,
2000).
More recently autofluorescence (AF), originating
from lipfuscin (Delori et al., 1995), has been used to
estimate MPOD. The advantage this technique offers is
an estimate of a single-pass absorption through the MP,
because the fluorescence can be recorded at wavelengths
that are not absorbed by MP. The disadvantage is the
low signal that is recorded and the specialized equipment that is required. The method can be implemented
with (Robson et al., 2003) or without imaging the retina
(Delori et al., 2001). Delori et al. (2001) found that the
AF estimates of MPOD more closely match those
derived from psychophysics (HFP) than the estimates
derived from fundus reflectometry. The imaging study
by Robson et al. allowed additional information
concerning the spatial profile of MP to be obtained. It
should also be noted that AF generated more reproducible results than fundus reflectometry and HFP
(Delori et al., 2001).
One recent development has been to use resonant
Raman spectroscopy to evaluate MPOD in vivo (Gellermann et al., 2002). This technique has generated
promising results but further work is required before the
method can be properly compared with established
procedures.
The objective techniques we have described certainly
offer advantages over psychophysical procedures: most
particularly, the subject does not need to maintain
fixation nor undergo extensive periods of observations.
It is worth noting that the objective measures of MPOD
considerably underestimate those derived from psychophysics.
8. Factors affecting MPOD within normal populations
The presence of MP in different amounts in different
individuals is interesting, particularly if the MP has a
role in the continued health of the macula or for
maximization of foveal visual function. In this
section, we review the data available to date regarding
any systematic variations in MP density that there
may be in different physiological and geographical
situations.
8.1. Comparison between eyes
Hammond and Fuld (1992) performed a specific study
on both eyes of 10 subjects. Using HFP the subjects had
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their MPOD assessed for each eye. The results showed
that in all eyes the difference in MPOD was less than 0.1
log units. Interestingly, there was some variability in the
measurements made over a period of several days (i.e.
on some days the measurements showed a difference
between eyes, but on other days not) but overall, there
was no consistent difference between eyes.
Colour matching has also revealed the correlation
between the MPOD in one eye with that in the other
(Morland, 1992). The data derived in that study are
reproduced in Fig. 7. Differences between the values
obtained from the two eyes exceeded 0.1 log units in two
of the 12 subjects, but overall there was no significant
difference between the values obtained from the left and
right eyes for the group (paired t test, t ¼ 1:2; p > 0:05
(two-tailed)).
Two subjects were involved in the study by Landrum
et al. (1997) to assess the effect of an oral lutein
supplement. The MPOD was measured in both eyes and
in one subject the levels were essentially the same,
whereas in the other, the MPOD in the left eye was
consistently about 0.1 log unit greater than in the right
eye.
Beatty has also measured MPOD in both eyes in a
group of healthy subjects as part of a study for risk of
AMD (Beatty et al., 2001). The MP density was
0.28970.156 for the right eye and 0.29970.159 for the
left eye.
From these studies, it would appear that the MP
quantities present in the eyes of individuals are, in the
main, similar. This has important consequences for the
study of large numbers of eyes; data from right and left
eyes should not be pooled but treated separately.
545
8.2. Twin studies and genetics
Another important issue is to try to establish whether
MP levels in the retina are genetically determined. The
mechanisms of incorporation into retinal tissue are not
understood at present. The existence of a specific
transport mechanism seems likely, given the observation
that there are approximately 40 dietary carotenoids and
only two in the macula. It has been shown that the
macular carotenoids are bound to tubulin both in
bovine and human retinae (Bernstein et al., 1997).
A transport system requires the production of the
necessary apparatus and this requires the genetic code
for its manufacture.
Hammond et al. (1995) used HFP to measure the
MPOD in 10 pairs of monozygotic twins. Serum levels
of L and Z were measured using HPLC and dietary
intake assessed using a food frequency questionnaire.
This study found significant differences in MP levels in 5
out of the 10 pairs of twins. There was no relationship
between dietary intake of carotenoid or serum levels
with MPOD. It was shown, however, that the differences in MPOD in the 5 pairs were related to the
differences in intake of dietary fat, iron, linoleic acid and
fibre. The conclusions drawn were that the levels of MP
in the macula are not completely genetically determined
and that there are likely to be multiple factors involved
in the deposition of MP in the retina.
8.3. Gender differences
The possibility of a consistent difference in average
levels of MP between males and females has also been
MPOD in different eyes
1.0
Right Eye
0.8
0.6
0.4
0.2
0.0
0.0
0.2
0.4
0.6
0.8
1.0
Left Eye
Fig. 7. MPOD measured with a 1.33 field at 460 nm with a colour-matching technique (Ruddock, 1963). Data for the left eye are plotted against
data for the right eye. The straight line is the least-squares fit of the data with a line passing through the origin with the equation right=0.88 left
ðr ¼ 0:79Þ:
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investigated (Hammond et al., 1996a) (this was done as
part of the associative studies of MPOD with agerelated macular degeneration, following the observation
that AMD is more common in females).
Hammond et al. studied MPOD in a group of males
and females and found that males had MPOD 38%
higher than females. The study measured MPOD in 48
women and 40 men. Geographically, 45 were from New
Hampshire and 43 of the participants were from the
Boston area. The mean MPOD for the men was
0.3870.216 and for the women 0.2470.159. Another
study on a population from the Southwest USA on 79
men and 138 women gave mean MPOD for the men as
0.2470.15 log units and for the women 0.2170.12 (13%
lower in women).
Interestingly, a large study performed in the Netherlands using fundus reflectometry on 199 men and 236
women did not show any gender difference in MPOD
(Berendschot et al., 2002a). A study based on 280
volunteers from the Midwest USA also showed no
gender difference in MPOD (Ciulla et al., 2001).
From these studies, it does not appear clear whether
there is a systematic difference between MPOD in men
and women.
8.4. Age
The results of many studies will be included in this
section, some of which were designed to investigate the
variation of MPOD with age and others where age
analysis was performed as part of the study protocol,
even if age variation was not the primary aim of the
study. Unfortunately, in only one of these studies was
the testing longitudinal (Hammond et al., 1997c) and in
that study the data from only ten persons was available
over time span ranging from 1 to 16 years.
The remaining studies are cross-sectional in nature
and one is left making the assumption that if MPOD
shows an age-related change in a sufficient number of
people this represents a true measure of the tendency of
MPOD to change with age during an individuals’
lifetime. A prospective, truly long-term study would be
extremely difficult to perform for obvious reasons and
although the above assumption is likely to be flawed the
results of large cross-sectional studies may be the closest
we can get to answering the question of whether MPOD
does change with age. Disappointingly, the results to
date on studies of age dependence in adults have been
variable. Below we review the evidence for and against
age dependence in adults.
The HPLC studies by Bone et al. (1988) and by
Handelman et al. (1988) on adult maculae and retinae
did not show any age-related decline in MPOD. Using
two-colour fundus photography, Chen et al. (2001)
separated the results of 54 subjects into three age
categories with mean ages 24.8, 40.2 and 67.5 years.
They did not find any association of MPOD with age,
but did find a change in the spatial profile of the
distribution, with older subjects having a broader
distribution. In a paper comparing the measurement of
MPOD using an autofluorescence technique with both
fundus reflectometry and HFP, a total of 159 subjects
were studied and no relationship with age was found
(Delori et al., 2001).
The findings of the more objective methods above
agree with those of several psychophysical studies by
Ruddock (1965), Werner et al. (1987), Hammond’s
group (Hammond and Caruso-Avery, 2000; Hammond
et al., 1997c), Mellerio et al. (2002) and Davies and
Morland (2002). Of note, only one of these studies
contains longitudinal data (Hammond et al., 1997c) and
this was in ten subjects. The data were collated from
studies performed in several laboratories, using the same
stimulus conditions. Importantly, no age-related change
was seen in any of the subjects. The study by Mellerio
used HFP and the MPOD of 124 eyes of 124 subjects
was measured. Davies and Morland (2002) used colour
matching and measured MPOD in 34 control subjects,
again finding no age-related change, although investigation of MPOD as a function of age was not the main
aim of the study. Two hundred and seventy-one subjects
took part in the study by Ciulla in the Midwest United
States and a multivariate analysis was performed on a
total of 58 variables (Ciulla et al., 2001). The outcome of
this analysis did not show age as a significant factor.
Age-related decline in MPOD has, however, been
reported in other studies. Using resonance Raman
spectroscopy, Bernstein et al. (2002) measured MP
levels in 220 eyes of 138 subjects in the age range 21–
84 years. The results clearly show an age-related decline
in MP, measured as spectrometer counts. Calibration
showed a linear relationship of spectrometer counts with
MP optical density. The results from left and right eyes
were not separated, however. Beatty et al. (2001)
measured MPOD in 46 healthy controls, aged 21–81
years. They found a small age-related decline of
approximately –0.05 log units per decade (this figure
was calculated by us from the best fit line given in Beatty
et al., Fig. 4). Another psychophysical study by
Hammond in the Southwest United States, as a part
of the studies investigating the geographical variation of
MPOD in North America, also showed a small but
significant decline in MPOD with age of 0.01 log units
per decade (Hammond and Caruso-Avery, 2000). There
is a large spread in the data and the correlation
coefficient was low ðr ¼ 0:14Þ:
Drawing conclusions from these studies is difficult, as
different techniques were used, different populations
studied and different levels of analysis were performed
on the data. We feel at present that the data available
indicate that there is probably no cross-sectional change
in MPOD with age once adult levels have been achieved.
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However, it is apparent that further study is required
across all age ranges in a large number of subjects in
order to provide a more definitive answer to the
question of age dependence.
8.4.1. Children and young adults
Recently, Bour et al. (2002) published a technique of
two wavelength fundus photography to measure MPOD
in children. Twenty-three subjects were studied, in the
age range 6–20 years. The mean value of MP found was
0.1370.04 log units. The results are lower than those
obtained using a similar technique in older subjects
published in two other studies (Chen et al., 2001; Elsner
et al., 1998). These latter studies used different calculations on the images and there could therefore be
systematic differences between them, which make direct
comparison of the results difficult.
Bour et al. (2002) did not present any results of the
use of the technique to measure MPOD in adults, nor
were any psychophysical investigations to measure MP
performed on the young subjects. This lack of comparison leaves some doubt as to whether the low values
observed represented a true finding or an underestimation of the MPOD because of the method used.
8.4.2. Prenatal, neonatal and infants
In his review from 1981, Nussbaum noted that at that
time there were no data regarding the presence of MPs
in the neonate or infant. In the absence of any further
information, it would also seem reasonable to hypothesize that MP may play a role in the development of the
macula lutea and that the levels seen in adulthood
simply represent an embryonic remnant. Levels of lutein
in foetal cord blood are significantly lower than those
found in maternal peripheral blood (Yeum et al., 1998).
These samples were obtained at the time of delivery and
may not represent the situation during gestation. It is
tempting to deduce that the transfer of macular
carotenoids across the placenta is low or absent. It has
also been shown that levels of lutein are two to three
times higher than b-carotene in human breast milk,
whilst their blood levels are nearly the same (Jewell et al.,
2001). Firm evidence about the presence of MP in the
retinae of premature infants (17–22 weeks gestation) was
provided by Bone et al. (1988), where retinae were
dissected and carotenoid levels were measured by
HPLC. The delicate nature of the tissues did not allow
dissection of the macula specifically, and whole retinae
were analysed. Both L and Z were detected in such
retinae, with abundance comparable to that of adults on
the basis of mass per unit area. However, in the prenatal
retinae, no yellow spot was seen in the macula region. In
the postnatal infants, a yellow spot was visible after B6
months of age.
In neonates and infants up to the age of 2 years, both
L and Z were detected in a disc of tissue of diameter
547
4.7 mm taken from the macula. Bone et al. (1988) found
in infant retinas below the age of 2 years that lutein was
the major pigment, whereas in older subjects zeaxanthin
was predominant. The mean ratio of L:Z in the infants
was 1.4470.16 for those less that 2 years of age and fell
to 0.7770.20 for those greater than 2 years. The retinal
distribution of MP in the neonatal and infant macula
was not studied. From this study it is apparent that MP
is present in the retina before birth; however, unanswered questions about the accumulation and concentration of MP in the macula itself remain. As a final
point, the causes of death in these unfortunate infants
were not given in the study and it remains a possibility
that the retinae analysed were not representative of the
true situation in vivo.
Handelman et al. (1988) measured MP in some young
retinae using HPLC and again found that L and Z were
present in the eyes of a 1-week-old neonate and also a 2month old. Levels were similar to those found in some
young adults, but older adults had higher levels in whole
retinal specimens. No maculae from infants were
studied.
8.5. MPOD in different populations
MP density is known to vary widely between different
individuals and there are many studies in the literature
from different centres across the world. In this section,
we review the findings from different centres, to
investigate whether there may be systematic differences
in MP density in different populations across the world.
It could be hypothesized that differences in race, diet,
sunlight exposure and other environmental factors
might give different populations different average MP
densities.
The majority of the work to date on MP density has
been performed in the United States, the United
Kingdom and the Netherlands. To our knowledge,
there are no studies of MP density in people living in
Eastern Europe, Scandanavia, France, Germany, Italy,
Greece, Turkey, the Middle East, India, most other
Asian countries, South and Central America or Africa.
In Section 7 above, we described a variety of different
methods that are available for measuring MPOD and
noted that there can be systematic differences in MPOD
measured in the same individuals using different
techniques. (e.g. photographic techniques tend to underestimate MPOD in comparison to psychophysical
methods). At present, there is no agreed ‘Gold Standard’
method for measuring the in vivo correlate of MP
density. Review of the work performed to date in
different centres around the world shows that a wide
range of the available techniques have been used. There
is also methodological variation within the same general
measurement technique. For example, many studies
have employed HFP to measure MPOD, but the studies
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have used different target sizes, stimulus wavelengths,
background stimulus conditions and locations for the
eccentric target position. All of these stimulus features
are likely to have an impact on estimates of MPOD and
if comparisons are to be made between studies with
differing stimulus features then (at the very least)
assumptions have to be made concerning the underlying
spatial and spectral properties of macular pigmentation.
It is very difficult, therefore, to evaluate differences
between the means of samples drawn from populations
in different geographical regions unless identical techniques have been used.
We tabulate the results of studies performed on
different populations across the world and also make
note of the numbers of subjects assessed. We also draw
the reader’s attention to the methods used in each study
and note that they frequently differ and that stimulus
attributes in studies that share the same methodology
also differ (Table 3). There are a few studies that have
evaluated MPOD in different geographical regions using
comparable methods and we review these below.
The earliest study to assess population differences was
conducted by Bone and Sparrock (1971). HFP was used
to measure MPOD in 49 subjects and the analysis
directed towards comparison of MPOD across different
racial groups, age, colour of iris and also hair. The
spectral absorption characteristics were obtained for the
MP from 400 to 580 nm in 10 nm steps. In this study,
there was no systematic difference in peak MP density at
460 nm in the different racial groups, or with age or iris
colour. There was no information reported about
whether these subjects lived in the same geographical
area or were recruited from different locations. The only
significant association was a high MPOD in subjects
with red hair.
Some of the studies conducted in the US by
Hammond and co-workers have used the same technique, which does allow for valid comparison of data
obtained in the different geographical locations within
the US. A large study was performed by Ciulla et al.
(2001), using HFP to assess MPOD in a group of 280
healthy adults from the Indianapolis area in the
Midwestern US. Overall, the mean MPOD was 0.21
(SD 0.13) log units. Using the same technique and
stimulus attributes, Hammond and Caruso-Avery
(2000) measured MPOD in 217 men and women from
Pheonix, Arizona. The mean MPOD in this group was
0.22 (SD 0.13) log units and thus it appears that the
means of samples taken from mid-western and Southwestern populations are no different. The mean MPOD
seems low in both studies compared to other evaluations
of MPOD using HFP and is likely to be a result of the
extrafoveal flicker measurement was made at 4
eccentricity.
In summary, we find very little data to date that allow
insight into whether any systematic variation exists in
Table 3
Mean MPOD obtained for samples of 30 or more normal subjects
Author and year
Location
Method
Davies and Morland (this
article)
Bone and Sparrock (1971)
Beatty et al. (2001)
UK
Colour Matching
30
Jamaica
UK
HFP
HFP
49
46
Davies and Morland (2002)
Mellerio et al. (2002)
Broekmans et al. (2002)
Berendschot et al. (2002)
Hammond et al. (1996c)
Hammond et al. (1996a)
UK
UK
Netherlands
Netherlands
Boston, USA
Boston and New
Hampshire, USA
Colour matching
HFP
Spectral reflectance
Spectral reflectance
HFP
HFP
Hammond et al. (1996b)
Boston and New
Hampshire, USA
HFP
Ciulla et al. (2001)
Hammond and Caruso-Avery
(2000)
Chang et al. (2002)
Mid West USA
South West USA
HFPz
HFPz
Taiwan
Fundus reflectometry
Number of
subjects
Subgroup
MPOD
(log units)
SD
0.56
Males
0.49–0.69
0.29
0.30
0.32
0.41
0.33
0.33
0.34
0.38
0.16
0.16
0.24
0.16
0.15
0.16
0.15
0.22
Females
Blue/Grey
0.24
0.25
0.16
0.20
Green/Hazel
Brown/Black
280
217
0.32
0.38
0.21
0.22
0.15
0.24
0.13
0.13
55
0.23
0.07
34
124
376
289
30
88
95
OD
OS
Those studies marked with the symbol z can be compared with each other on the basis that the technique and stimulus attributes are the same.
Although other studies have employed the same technique, the stimulus attributes vary; so direct comparison of mean MPOD values is not possible.
The column labelled ‘Subgroup’ indicates which subgroups were investigated in studies that differentiated their normal subjects into different
categories.
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MPOD in different populations across the world. It
would require significant dedication and time to
investigate this, but it is entirely possible. Would such
an investigation be worthwhile? We believe that it would
be; the results of a large study using one or more
methods would indicate if some populations exposed to
highly different environments have different MP levels.
This in turn may indicate whether MP does indeed play
an active physiological role in humans.
8.6. Other factors influencing MPOD in normal
populations
8.6.1. Tobacco smoking
Levels of MP were first measured in smokers in
comparison with non-smokers by Hammond et al.
(1996c). Increased oxidative stress may be one of the
causative factors in several smoking-related diseases and
the study was performed to investigate the possible
relationship between smoking and macular carotenoid
levels. Smoking has been shown to significantly decrease
levels of carotenoids in serum and also to increase the
risk of neovascular age-related maculopathy (Klein
et al., 1993; Paetkau et al., 1978; Snodderly, 1995;
Vingerling et al., 1996).
Thirty-four smokers and 34 non-smokers were
studied. There were no differences in age, weight,
different skin tones, hair or iris colour between the
two groups. The results showed a significantly lower
MPOD in the smokers in comparison with non-smokers
(mean MPOD 0.16, SD 0.12 vs. 0.34, SD 0.15;
po0:0001). The difference in MPOD could not be
explained by differences in dietary intake of carotenoids
assessed by questionnaire. Also, the reduction in MPOD
in the smokers showed an inverse relationship with the
number of cigarettes smoked; smoking more than 25
cigarettes per day having a pronounced effect on
MPOD.
Following this study, other studies have included
smoking as part of a sub-analysis. In the Southwest
USA, smoking has been identified with a low MP
density (Hammond and Caruso-Avery, 2000), but no
significant relationship was found in a Midwest population (Ciulla et al., 2001).
As a part of the validation of different measurement
techniques, Delori et al. (2001) assessed MPOD in 27
smokers compared with 102 non-smokers. The results
showed a small but statistically significant reduction in
MPOD measured using lipofuscin autofluorescence
(mean 0.4070.15 vs. 0.4970.16 log units). No difference was noted between the two groups when MPOD
was measured using fundus reflectance, however. Details
of the amount of tobacco consumed were not given.
Using HFP alone, Mellerio et al. (2002) also noted a
smoking-related reduction in pigment density.
549
These studies indicate that the MPOD is likely to be
reduced by tobacco smoking, most particularly in heavy
consumption (>25 cigarettes per day). The implications
of this finding are discussed more fully in the context of
age-related maculopathy.
8.6.2. Iris colour
A study published in 1996 was specifically designed to
investigate whether there is an association between iris
colour and MPOD (Hammond et al., 1996b). This study
was performed based on the finding that a dark iris was
associated with reduced risk of AMD (Hyman et al.,
1983; Weiter et al., 1985). It is known that eyes with
light-coloured irides transmit significantly more light
than those with dark irides (van den Berg et al., 1991)
and the hypothesis was suggested that MP levels are
lower in eyes with light iris colour because of increased
oxidative stress due to light exposure.
Ninety-five non-smokers participated in the study and
iris colour was classified into one of three groups, blue
or grey, green or hazel and brown or black. There was a
reasonably even distribution of gender, race and age
between the groups. MPOD was measured using HFP
and serum L and Z levels by HPLC. There was no
significant difference between the serum levels of L and
Z in the three groups, nor of dietary carotenoid intake.
There was a significant difference in MPOD between
those with blue/grey and brown/black iris colour, but no
difference between those with green/hazel and brown/
black irides or blue/grey and green/hazel irides. There
was no difference in MPOD with respect to gender.
8.6.3. Lens density
The presence of carotenoids in the crystalline lens has
been documented by Yeum et al. (1995, 1999) and
Bernstein et al. (2001). Hammond measured both
MPOD using HFP and lens optical density using
scotopic sensitivity in a total of 41 subjects (Hammond
et al., 1997b). They found that there was no relationship
between MPOD and lens OD for subjects aged 24–36
years. A significant relationship was found, however, for
middle-aged subjects (range 48–66 years) and even more
so for older subjects (age range 67–82 years).
The finding of a lower MP level in the eyes with lenses
that are more optically dense was used to support the
hypothesis that retinal MP levels act as markers for
lenticular MP levels, and that lenticular carotenoids act
to protect the lens from age-related increase in lens
density.
We can suggest an alternative hypothesis for the
above finding. Retinae screened from high-energy light
(by the lens) do not need to accumulate MP as a
protection against its effects. As the mechanisms
controlling incorporation of MP into the retina are
not understood, it does not seem implausible to suggest
that MP levels may be controlled partly by the amount
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of incident light. This hypothesis could also be employed
to explain our findings in patients with diabetes (Davies
and Morland, 2002) (see later).
8.6.4. Obesity
Lutein and zeaxanthin are stored in greatest proportion in body fat and obesity is known to be a risk factor
for age-relatel macular degeneration (AMD) (Schaumberg et al., 2001). To investigate whether total body fat
and body mass index (BMI) influence the MPOD,
Hammond et al. (2002) undertook a study of 680
individuals. MPOD was measured using HFP, and data
were collected on BMI and body fat percentage. There
was a significant negative relationship between MPOD
and both BMI and percentage body fat. It was made
note of that this relationship was mainly the result of the
significantly lower pigment density (21%) in subjects
with a BMI greater than 29.
9. Macular pigmentation in disease
9.1. AMD
AMD is the commonest form of blindness in the
developed world. The mechanisms underlying its pathogenesis are not fully elucidated and treatment options
once the disease is established are limited. There is
evidence that photocoagulative and, more recently
photodynamic laser, treatment (‘Report’ 2000; Barbazetto et al., 2003) is beneficial in some subgroups and in
other subgroups there is now evidence that anti-oxidants
have a protective effect (ARED report no. 8, 2001).
Although it is not within the scope of this article to
investigate the pathogenesis of AMD in detail, here we
review the data relevant to AMD and the carotenoids.
There is epidemiological evidence that people at a
higher risk of AMD have lower intake and serum levels
of L and Z (EDCC). Other risk factors for AMD include
being female gender, smoking tobacco (Klein et al.,
1993; Paetkau et al., 1978; Snodderly, 1995; Vingerling
et al., 1996), having a light iris colour (Sandberg et al.,
1994), and obesity (Schaumberg et al., 2001). In a search
for a common thread with these risk factors, Hammond
et al. have conducted a series of studies measuring the
MPOD as a function of the above parameters in
isolation (see Section 8 for discussion of these results).
All of the studies found a relationship with low MP
levels in the variables studied. This finding, coupled with
the knowledge that MP can act as antioxidants, that the
macula is a site with great potential for oxidative stress
(see later) and in particular the hypothesized role that
oxidative stress plays in the pathogenesis of AMD
(Beatty et al., 2000a) may indicate that the MPs could
have a role in protecting the macula from the changes
that ultimately lead to AMD. Another study noted that
the fovea can be preserved and the perifovea affected by
degeneration (Weiter et al., 1988), suggesting that the
fovea is protected by the high density of MP located
there. (From the evolutionary point of view, we wonder
what mechanism could have driven the accumulation of
a substance that protects the animal from a disease that
only occurs after the age of reproduction and rearing.)
Below we review the results of studies that have been
designed to measure the MPOD in patients with or at
risk of AMD. Beatty et al. (2001) used HFP to measure
MPOD in a small group of 9 subjects at high risk of agerelated macular degeneration and in a group of 46
healthy volunteers in the age range 21–81 years.
The eyes at risk of AMD were chosen from 9 patients
who had advanced neovascular AMD in the fellow eye
and yet no macular abnormality in the study eye. This
prerequisite for the study was necessary as the study
used HFP and thus requires the assumption of normal
receptoral function and comparable spectral sensitivity
foveally and extrafoveally. However, it should be
pointed out that the patients in this category form an
unusual group. AMD is predominantly a bilateral
condition and most patients with advanced neovascular
change in one eye have a degree of AMD in the fellow
eye. This is borne out in the small number of ‘high-risk’
eyes studied. It is also known that fellow eyes with no
macular abnormality have the lowest risk of progression
to neovascular AMD in comparison with fellow eyes
with soft drusen, focal hyperpigmentation and systemic
hypertension (Bressler, 2001). The results showed that
the high-risk eyes had a significantly lower MPOD in
comparison with healthy subjects (0.14770.144 log
units vs 0.33170.206 log units, p ¼ 0:015; Wilcoxon
ranked sum test) after matching for age, gender, iris
colour, smoking habits and lens density.
Using Raman spectroscopy, Bernstein et al. measured
MPOD in 220 eyes of 18 normals and 93 eyes of 68
patients with AMD (Bernstein and Gellermann, 2002).
The results from both right and left eyes were pooled.
The mean spectrometer counts were given from a subset
of the control group (those greater than 60 years) and in
AMD patients taking supplements containing X4 mg
lutein and those whose supplemental intake of lutein
was zero or less than 4 mg/day. The counts were
2197134 for controls, 2127169 for AMD taking lutein
and 1487147 for the no-supplement group. This result
indicates that the patients with AMD not taking any
lutein supplement had a significantly lower MP level
(32%) than age-similar normals or those taking lutein.
A calibration figure is given in the paper for spectrometer counts as a function of MPOD (based on a
standard sample). Using this graph we find that the
levels of MPOD in all groups are very low—a count of
200 gives an MPOD of slightly less that 0.05 log units.
Using spectral fundus reflectance, Berendschot et al.
(2002b) measured MPOD in a group of 289 eyes with no
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AMD and 146 eyes with AMD (single eye per subject).
All participants were X55 years of age. It was made
note of that none of the subjects were using lutein
supplementation at the time of measurement. The
MPOD was measured using a specific optical model
(van de Kraats et al., 1996). The effect of drusen was
ignored in the model. However, the results suggested a
spectrally flat reflectance from the drusen, which would
not affect the determination of MPOD. Interestingly,
the results of this study showed a mean MPOD in the
control group of 0.3370.15 and 0.3370.16 in eyes with
any stage of AMD (analysis used a general linear model
of age and AMD stage).
9.2. Diabetes
We used colour matching to assess MPOD (Ruddock,
1963) in a group of 34 patients with diabetes and 34
healthy controls (Davies and Morland, 2002). The
Wright tristimulus colorimeter was used (Wright,
1939), with a test field consisting of 490 nm desaturated
with 650 nm and matching fields of 460 and 530 nm,
desaturated with 650 nm. The field was a bipartite
square of angular dimension 1 2000 and the extrafoveal
match made at 5 of eccentricity. In this study, the mean
MPOD of the normal subjects was 0.3270.24 log units
and of the diabetic subjects 0.1370.20 log units ðp ¼
0:0015Þ: The MOPD showed a negative relationship
with increasing grade of maculopathy. We modelled the
change that would be expected in measurement of
MPOD based on differential receptor dysfunction at
foveal and extrafoveal locations (see above) and found
that the effect on MPOD measurement was small and
could not explain the results observed. The findings of
this study are interesting, as it can be argued that the
macula in diabetes is prone to greater oxidative stress
than without and that the development of anatomical
changes may be a good marker of the severity of the
retinal microvascular disease.
To date, we are not aware of any other studies that
have measured MPOD in patients with diabetes.
9.3. Other conditions
9.3.1. Albinism
The study of MPOD in human albinism has revealed
that negligible pigment can be detected in such
individuals. This has been revealed by fundus photography by Abadi and Cox (1992). In this case, an
objective measurement is particularly useful, because
behavioural tests applied to subjects with albinism will
be prone to underestimate pigment density because of
the nystagmus that these subjects frequently display.
Behavioural measurements in subjects with albinism and
very little nystagmus undertaken in our laboratory have
confirmed the findings of Abadi and Cox (1992) with a
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group of eight oculocutaneous albinos having a mean
density of 0.04 that was not significantly different from
zero. It is interesting that individuals with very low levels
of ocular melanin also have negligible levels of the MPs
that are derived from dietary intake of carotenoids. It
remains to be seen if the retinae of such subjects are
devoid of the pigments, or whether measurements are
incapable of finding the concentration of them in the
macula that is typical in normal subjects. It is certain
that subjects with albinism do not have a normally
developed macula, so perhaps this structural abnormality prevents the appropriate accumulation of the
relevant pigmentation in one area of the retina. A
comparison of MP levels in patients with aniridia may
be fruitful in terms of disambiguating the relative role of
hypopigmentation and abnormal foveal development on
MPOD.
9.3.2. Choroideraemia
MP levels and macular function were measured in a
group of patients with choroideraemia (Duncan et al.,
2002). This inherited disease causes progressive degeneration of photoreceptors, RPE and choroid. Thirteen
patients with chorioderaemia and 40 controls took part.
Lutein supplement in a dose of 20 mg/day was taken for
a period of 6 months. The MP levels prior to supplement
were not different in the two groups and both showed a
rise in MPOD with the supplement. The patients with
chorioderaemia had reduced central rod and cone
function, as measured with two-colour dark adapted
sensitivity. There was no change in the retinal sensitivity
after the supplementation period. This study concluded
that there was no short-term benefit of oral supplement
in this group. The need for a longer term study on the
effects of oral MP supplement was noted.
9.3.3. Retinitis pigmentosa
MPOD was measured using HFP in patients with RP
and Usher’s syndrome (Aleman et al., 2001). The
MPOD in the patients with RP was similar to that of
normals and bore the same relationship with markers of
low MP in normals (i.e. lower in females, smokers and
persons with light-coloured irides). Oral supplementation with lutein resulted in a rise in MPOD in only half
of the patients and there was no detectable change in
central visual function.
10. Dietary supplement of macular pigmentation
The fact that the MPs are entirely of dietary origin
and the thought that they may play some role in
protecting the macula has encouraged researchers to
investigate the effect that increasing consumption of L &
Z has on the MP density in the eye. Landrum et al.
(1997) performed a study of lutein supplementation on
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two subjects for a period of 140 days and followed them
for 1 year. Lutein esters were given (derived from
marigolds) at a dose of 30 mg/day. MPOD was
measured using HFP 4–5 times per week and serum
concentrations determined by HPLC. For both subjects
there was a clear rise in MPOD for both eyes (39% for
one subject and 21% for the other), which reached a
plateau around 50 days after cessation of supplement.
This rise in MPOD was maintained for the duration of
measurement.
In another study, Hammond et al. (1997a) used
dietary supplement with spinach and sweetcorn to give
an intake of approximately 10 mg of lutein and 0.7 mg
zeaxanthin. Thirteen persons participated in the study,
of which 12 adhered to the dietary supplement for 15
weeks. Serial measurements were made using HFP and
serum levels monitored using HPLC. Analysis of results
indicated three different responses to the supplement of
spinach and sweetcorn. The first group (8/11) had
increases of both serum and retinal levels of MP. The
second (2/11) had a serum, but not a retinal response,
and one subject showed neither a serum nor a retinal
response. The two other subjects were given sweetcorn
only (i.e. zeaxanthin supplement only) and one showed a
significant rise in MPOD, whilst the other did not.
Two different MP measurement techniques were used
by Berendschot et al. (2000) to assess the effect of lutein
supplement on eight male volunteers. A 10 mg daily dose
of lutein was taken and MPOD measured using log
reflectance maps obtained by a scanning laser ophthalmoscope and also using spectral reflectance. All subjects
showed a linear rise in MPOD with a mean 4-week
increase of 5.3%. This study is the first to use an
objective technique to measure the effect of lutein
supplement on MPOD.
More recently, a further supplement study reported
the results of HFP measurements on 38 subjects, with
different dosages of lutein and zeaxanthin. This allowed
the estimation of the serum response with respect to
L+Z dose and also the MPOD increase with respect to
the serum response (Bone et al., 2003). Overall, the
response looked linear, with two-thirds of the variance
of serum levels attributable to oral dose and one-third of
the variance of MPOD attributable to serum levels.
These studies clearly indicate that increasing dietary
consumption of the macular carotenoids can raise both
the serum and retinal levels of MP. Although the
response is variable across different individuals, there
exists the possibility that oral supplement of MP could
be used for either visual or therapeutic purposes.
11. Putative roles for MP
The presence of two selected carotenoids in the
macula is generally assumed to imply that they serve
some useful function for the animal. From an evolutionary perspective, it could be argued that the MP
confers survival advantage to the animal and should do
so within its reproductive lifespan. Two roles for MP
(not mutually exclusive) have been proposed for their
function, and in this section we review the evidence for
both.
11.1. Role in improving visual function
Improving visual function would seem to us to be the
most logical role for MP. As the macula is specialized
for high spatial resolution and for colour vision, it
would seem that MP could be involved in these
processes.
11.1.1. Chromatic aberration
The eye suffers from a relatively large amount of
chromatic aberration. Longitudinal chromatic aberration (LCA) results from the dispersion characteristics of
the ocular media. With the preferred accommodation on
the wavelengths of peak sensitivity (550 nm), the SW
light is focused anterior and the LW light posterior to
the retina. This results in a penumbra on the retina of
both blue and red light. The amount of LCA in the eye
has been measured experimentally (Bedford and Wysecki, 1957) and is relatively constant across individuals
(Fig. 8). The range is approximately 2.1 D from 400 to
700 nm. With the eye accommodated on 550 nm light,
light of 460 nm has a defocus of approximately 1.2 D.
Due to the dispersion, the LCA for longer wavelengths
is less, being around +0.5 D for 650 nm light.
Transverse chromatic aberration (TCA) also affects
the eye, and its effects have been studied in less detail.
TCA results in LW light being deviated less than SW
light. This has the effect of giving a red blur at the edge
of the image. Taken together and when viewing white
light, LCA and TCA would result in the presence of a
purple penumbra to the image.
0.5
Chromatic difference of refraction (D)
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0.0
-0.5
-1.0
Data from Bedford and Wyzsecki (1957)
-1.5
-2.0
350
400
450
500
550
600
650
700
Wavelength (nm)
Fig. 8. Longitudinal chromatic aberration of the eye. Data from
Wysecki and Stiles (1982) Table 1 (2.4.3) (originally reported by
Bedford and Wysecki, 1957).
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In 1866, Schultze proposed a role for MP of
absorbing sufficient SW light to reduce the blur in the
retinal image resulting from LCA. This was investigated
by Reading and Weale (1974), who showed that a filter
required to reduce radiance of the SW blur circle to a
subthreshold value covered a spectral range similar to
that of the MP.
This led to the hypothesis that the role of MP is to
reduce SW chromatic blur and thus to enhance spatial
vision. In their article in this journal, Wooten and
Hammond (2002) have termed this the ‘Acuity Hypothesis’. They note that there have been many trials of the
use of additional filters to enhance spatial vision. When
measuring high-contrast acuity, there is no proven
benefit and even when considering contrast sensitivity,
some studies have produced positive results and others
have found no effect. Overall, all studies have shown
that there are wide-ranging effects across different
individuals, some experiencing visual improvement,
others no change and others detriment to their vision
(Kelly et al., 1984; Wolffsohn et al., 2000). Amazingly
enough, no study has measured MPOD in the participants and correlated this with any improvement in
visual function with the use of a supplemental filter. A
hypothesis to explain the wide variation of response to
supplemental filters is that those subjects with high MP
levels would experience little or no visual enhancement,
whilst those with lower pigment levels may experience a
greater effect.
This does, however, raise the important issue of
why the MP levels are so variable across individuals.
If MP does indeed improve vision by removing the
effect of LCA, why are MP levels not uniformly high
(or controlled to lie within a narrow range), particularly as the effects of LCA are consistent across
individuals?
It has recently been suggested that retinal image
quality is in fact relatively independent of wavelength
(McLellan et al., 2002) and that the quality of the image
for shorter wavelengths is not degraded by LCA. This
study used a spatially resolved refractometer (Marcos
et al., 1999) to measure wavefront aberration data as a
function of wavelength in three individuals. Wave
aberration data were used to calculate the modulation
transfer function (MTF) of the eye and this was used as
the metric of image quality.
Aberrations were measured at six wavelengths (450,
490, 530, 570 and 650 nm). The area under the MTF was
examined as a function of wavelength and compared
with a theoretical MTF for a model eye with LCA only.
The results showed a relatively flat function for all three
subjects, whereas for the model eye the MTF area
decreased significantly as wavelength deviated from the
optimal focus of 550 nm. It should be noted, however,
that the subjects’ pupils were dilated with 0.5%
tropicamide only. At this concentration, tropicamide
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will allow some pupillary dilatation but is unlikely to
paralyse accommodation. It is possible that in the course
of the measurements the subjects were accommodating
onto the lights at the different wavelengths used and
thus reduced any blur from LCA, artificially improving
the MTF. An optical channel was included in the system
with a background of text to give an accommodative
target but this may not provide hard control of
accommodative state. To allow a formal assessment of
MTF as a function of wavelength in the presence of
LCA, it would be best that the subjects undergo
complete cycloplegia with several drops of cyclopentolate or indeed atropine. Also, the study was conducted
with a pupil diameter of 6 mm and at a relatively low
luminance of 100 cd/m2. It may not be safe to generalize
the results to the smaller pupil that occurs in natural
light of significantly higher luminance. Perhaps firmer
evidence questioning the role of MP in this context
could be attained by a more comprehensive experiment
with subjects under cycloplegia and measurements made
over a wider range of pupil sizes.
As a final point, in an earlier study by the same group
(Marcos et al., 1999), the analysis of optical quality with
respect to wavelength was presented in three ways:
volume under the MTF, RMS wavefront error and the
Strehl ratio. Interestingly, the volume under the MTF
for one subject decreased with increasing wavelength
(even when truncated for spatial frequencies >100 cpd).
The RMS value was relatively flat with respect to
wavelength (RMS indicates the change in phase of the
pupil function but does not include diffraction effects).
The Strehl ratio, however, increased significantly with
increasing wavelength. These different measures gave
optical quality metrics that were different for the same
individual and one must be careful about the interpretation.
In summary, we note that the evidence for and against
this role for MP is associative only and that further
study needs to be undertaken to confirm or refute the
hypothesis that MP improves visual function by
reducing the effect of chromatic aberration.
11.1.2. Visibility
Wooten and Hammond (2002) propose another
hypothesis of how MP may improve our vision. The
physics of light scatter is such that SW light is scattered
more than longer wavelengths both by air molecules
(Rayleigh scatter model) and by larger particles in the
atmosphere (haze aerosol, treated with the Mie scatter
model). Both of these lead to the blue colour of the sky
and the blue haze attained by objects viewed in the
distance. They show that a yellow filter can increase the
visibility of a target viewed against a background by
reducing the blue of the background. At the same time,
the spectral energy of any target is reduced in the shorter
wavelengths by scatter. Hence, the overall effect of a
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yellow filter will be to reduce the luminance of the
background with respect to the target, increasing its
contrast.
Further calculation showed that the visibility range of
an object seen at 10 km with an MPOD of 0.0 is
increased to 11.9 km with MPOD of 0.5 log units. The
corollary of this is that in the presence of MP, nearer
objects may become visible in the presence of MP that
would otherwise have remained sub-threshold. The
visibility hypothesis is attractive, but the gains in visual
function are rather modest.
11.2. MPs as antioxidants
The macula (and indeed the whole retina) is a very
delicate tissue and as with all neural tissues does not
have the ability to regenerate after damage. The retina
serves a vital role for the animal as the transducer of the
dominant sensory organ. Also, the retina is the most
metabolically active tissue in the body and the photoreceptor layer is maintained at a high oxygen tension
and contains a high concentration of polyunsaturated
fatty acid (Anderson et al., 1984). This environment sets
the scene for a tissue that is at risk of damage from two
sources. Firstly photic, from the incident and absorbed
light and secondly from internal processes, as both of
these mechanisms can lead to the release of reactive
oxygen species (superoxide anion, hydroxyl free radical
and hydroperoxyl radicals).
Ham (Ham et al., 1978) measured the damaging
effects of SW light on rhesus monkey retina. Depending
on exposure duration, the power required to result in
photic damage was 70–1000 times lower for 441 nm light
than for infrared light of 1064 nm (durations from 1 to
1000 s).
From these findings, it is apparent that the macula is
prone to irreversible damage from light of SW and it
therefore seems logical to propose that during evolution
protective mechanisms may have developed if such
damage were likely to occur during the reproductive age
of the animal. Thus, the second major hypothesized role
for the MP is as an antioxidant.
The MP could have two distinct roles in acting as a
protector of the macula. Firstly, given its location as a
prereceptoral filter (Snodderly et al., 1984a), to protect
the macula by reducing the amount of SW light reaching
the photoreceptor outer segments and secondly by
acting directly as a scavenger of reactive oxygen species
once liberated; either from light-induced damage or
from other internal mechanisms.
Considering the photic damage hypothesis, it is
interesting to note that MP is not concentrated in the
inferior retina. With blue-sky overhead, most of the SW
light will be imaged on the inferior retina and we do not
observe inferior retinal changes routinely in subjects
whose retinae are exposed to such influence. This
observation may go against the SW damage idea, except
to point out the counter argument that the macula is a
specialized area of the retina with the highest receptor
density and thus may be more prone to damage derived
from light exposure.
Here we examine the evidence that MP plays a
protective role for the preservation of the neural
elements that initiate our central vision. The mechanisms for retinal damage by reactive oxygen species have
been reviewed by Beatty (Beatty et al., 2000a) in
particular in relation to age-related macular degeneration and the interested reader is referred to this article.
The carotenoids as a family have clear antioxidant
properties and have been shown to react with singlet
oxygen, free radicals and also to prevent lipid peroxidation (Khachik et al., 1997). Also, oxidation products of
lutein and zeaxanthin have been identified in the retina
(Khachik et al., 1997).
The anatomical location of lutein and zeaxanthin in
the photoreceptor axons and in the inner plexiform layer
(Snodderly et al., 1984a) will shield photoreceptors from
SW light, but MP is not well located here to act as a
scavenger of free radicals and oxygen species released
near the photoreceptor outer segments. In view of this,
two studies have been performed to investigate whether
MP is also found associated with the receptoral
membranes (Rapp et al., 2000; Sommerburg et al.,
1999). It was found that lutein and zeaxanthin are both
present in the membranes of rod outer segments,
representing somewhere between 10% (Rapp et al.,
2000) and 25% (Sommerburg et al., 1999) of the total
retinal amount of MP. We are not aware of any studies
investigating specifically the presence of MP in cone
receptor outer segments.
The effect of MP as an antioxidant in the central
retina has been investigated in several studies. The
earliest was that by Haegerstrom-Portnoy (1988). In this
study, S- and L-cone sensitivities were measured across
the central retina in two groups of normals. In the older
group, there was a significant differential loss of S-cone
sensitivity across the retina compared to the younger
group, with increased S-cone sensitivity loss away from
the fovea. No such change was noted for the L-cones.
This study was interpreted as supporting the hypothesis
that MP protects the retina from light-induced damage.
Weiter et al. (1988) noted that there are some subjects in
whom the central fovea is preserved whilst the perifoveal
tissue degenerates. This again may imply a protective
role of the MP.
A more detailed study has been performed more
recently, where lens density, MPOD and Stiles p1
increment (related to S-cone) thresholds were measured
in a group of normals over a wide age range (Hammond
et al., 1998). The MPOD was measured using HFP and
the lens density using equivalent rhodopsin thresholds in
the dark-adapted eye. The Stiles p1 sensitivity was
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measured at 440 and 500 nm for a 1 foveal target. This
study found that there is a reduction of visual sensitivity
in the p 1 mechanism (corrected for both MPOD and
lens density) with increasing age. The decline in
sensitivity was correlated with MPOD, in that subjects
with a low MP (0–0.39) showed a significant age-related
reduction in sensitivity at 440 nm, whilst those with a
high MP density (0.40–0.97 log units) had no age-related
decline. Foveal sensitivity as a function of MPOD also
showed a correlation for the older subjects. Those aged
60–84 years had reduced foveal sensitivity as MPOD
declined, whereas the younger group (24–36 years) did
not show such a relationship. It seems reasonable to
conclude from this that higher levels of MP are
associated with maintenance of foveal S-cone function
in older persons, although this association may not be
causal.
Studies of oral supplementation with the macular
carotenoids are underway in patients with AMD. One
study investigated the effects of antioxidant supplement
on central retinal electrophysiological function in a
group of patients with AMD (Falsini et al., 2003). Here,
the supplement consisted of lutein (10 mg), vitamin E
(20 mg) and nicotinamide (18 mg). Focal electroretinograms were used to assess central retinal response and
the supplements were taken for a period of 180 days.
The results showed a significant increase in the ERG
amplitude in the patients with AMD taking the
supplement in comparison with those not taking it.
Furthermore, the values returned toward the pretreatment levels 180 days after cessation of supplement.
The control group showed a similar increase in
amplitude at 180 days. Unfortunately, in this study, no
measurements of serum or macular levels of MP were
made, which makes it impossible to draw conclusions
about the benefit the lutein supplement alone.
The Lutein Antioxidant Supplementation Trial (Richer et al., 2002) has been published in abstract form
only and consisted of lutein vs. lutein/antioxidants
supplements in a group of 90 elderly patients with
AMD. MPOD increased on average by 0.09 log units
(assessed by HFP), and there was a significant improvement in glare recovery, contrast sensitivity and both
near and distance acuities in both treatment groups. We
await the full publication of this and of other ongoing
trials into the effect of MP supplements with great
interest.
12. Outstanding issues
In this section, we present some issues that we believe
require investigation. The reader is also directed to the
previous section in which various potential avenues of
study that should shed light on the role of the MP are
also outlined.
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12.1. The importance of measuring the distribution or
peak MPOD
A very interesting result has recently come to light:
Peak MPOD measured for a small central region of the
fovea does not correlate well with the overall level of
pigmentation within the macula (Robson et al., 2003).
Given the increasing prominence of the hypothesis that
increased macular pigmentation may help prevent
AMD, it seems timely to question whether it is the
overall pigmentation of the macula or the peak MPOD
that plays the most significant protective role. It would
be valuable if future works explicitly evaluate which of
the two factors is most important in preserving visual
function and whether either or both can be modulated
by significantly by dietary supplement.
12.2. How should macular pigmentation be measured?
We reviewed psychophysical and objective methods
for evaluating macular pigmentation. In our view, there
is an increasing need to obtain as much information
about macular pigmentation as possible, not least
because of the issue we described above. We believe,
therefore, that the objective methods, which can readily
obtain estimates of peak MPOD and information on the
spatial distribution of MPs, should be preferred.
However, objective methods appear to provide consistently lower estimates of MPOD. This does not render
these methods invalid as long as they are reliable and
reproducible, which has been shown to be the case most
particularly for AF. Imaging methods also offer a great
advantage of not requiring the subject/patient to
undergo demanding observations. This is a key issue
for work that must be undertaken on elderly subjects,
those with macular pathology or ocular instability. It
must be noted, however, that light scatter in the ocular
media, which increases with age, could reduce the
accuracy of MPOD estimates with many objective
measures.
Although objective techniques are the most desirable
procedures to take forward, it must be recognized that
the methods are currently used in the few laboratories
with the necessary sophisticated equipment. This provides a reason why psychophysical procedures have been
the ones that most large-scale surveys of MPOD have
adopted. In our view, there is now a demand for an
objective imaging technique that can be readily implemented to measure MPOD and MP spatial distribution quickly and reliably. A modification to a standard
imaging ophthalmoscope would be most appropriate
and would allow ophthalmologists to readily acquire
data on macular pigmentation on large number of
subjects. Moreover, such a modified instrument would
enable the clinician to monitor the effect of dietary
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supplementation on macular pigmentation and perform
longitudinal studies.
12.3. Can macular pigmentation be modulated to serve a
protective role?
There appears to be converging evidence that macular
pigmentation can be enhanced by increased intake of L
and Z. In addition to evaluating the long-term effects of
L and Z, more data on how macular pigmentation may
be enhanced in disease is also required to add to the
encouraging preliminary results reviewed in this article.
12.4. Correlation and causality
There is evidence that suggests lower levels of macular
pigmentation in groups of subjects with AMD than in
groups of control subjects. From this finding, it is
tempting to draw the conclusion that low levels of
macular pigmentation may be a causative factor in
AMD. However, it is equally likely that lower levels of
macular pigmentation may result from neural loss in the
macula associated with AMD (i.e. a secondary effect).
In order to resolve the presence and direction of a causal
link between AMD and macular pigmentation, it is
necessary to perform longitudinal studies that can
follow subjects without AMD for sufficient time for
AMD to develop in some but not others. It may also be
of value to examine the relative proportions of oxidized
products of L and Z in the diseased retina.
12.5. Mechanisms of deposition of the MPs
An issue that remains truly outstanding is how L and
Z are deposited in the macula and how and if oxidized
products of L and Z are removed from the retina.
Addressing this issue will be a difficult task, but is
fundamental to understanding how macular pigments
may benefit individuals in health and disease. Genetic
factors that influence the population variation of
MPOD remain outstanding and work in this area may
yield better understanding of the fundamental processes
governing L and Z deposition in the macula.
13. Conclusions
The work over the past 50 years has identified and
characterized the MPs in health and in different disease
states. The relationship between dietary intake, serum
levels and MPOD is better understood. There has been
an increase in the number of techniques for in vivo
measurement of MP; each method has its own
advantages and disadvantages. We also note that the
multitude of methods used across the world makes
comparison of different works very difficult.
Perhaps, it is now time for the scientific community
interested in MP to agree on a preferred or standardized
technique for its measurement in humans. The design of
studies needs to move away from the gathering of
associative data and should aim towards understanding
the underlying mechanisms with the aim of addressing
causation of disease.
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